Current medical practice and health care policies require that clinically relevant specific binding analytes must be measured rapidly and economically. In order to perform an assay of an analyte that reacts in or with the result of a specific binding reaction, the prior art often resorted to the physical separation of the bound from the unbound labelled partner of the analyte and subsequent wash steps of the solid phase after a specific binding reaction has occurred. For example, R. Whitehead et al., in U.S. Pat. No. 4,544,088 disclose the use of collodial magnetic conjugates to separate radiolabelled antibody/antigen complexes. Other commercial embodiments use porous particles made from glass (see Bluestein et al. U.S. Pat. No. 4,780,423) or latex (see Zuk et al. U.S. Pat. No. 4,654,300). Incubation of particulate solid phase materials in liquids of high density to prevent settling and subsequent dilution to promote gravimetric settling as a method of separation was described by Baker et. al. in U.S. Pat. No. 4,656,143. In this case, dilution reduced the amount of unbound material physically associated with the settled solid phase.
In an attempt to eliminate separation and washing steps in binding assays, the art has used immiscible liquids or higher density media to keep particles suspended while a binding reaction occurs. First described in W.C. Werheiser and W. Bartley, Biochem. J. 66 pg 79 (1957), and later in M. Klingenberg and E. Pfaff, Methods in Enzymology, Vol X pg 680 (1967), and even more eloquently applied in R. McLilley et. al., Biochemica et Biophysica Acta 960 pg 259 (1977) chloroplasts are suspended in an aqueous medium over silicon oils during a C.sup.14 uptake (binding) reaction. To separate the bound C.sup.14 from unbound material the particulate chloroplasts were centrifuged out of the aqueous reaction medium, through the oil, and into a metabolic reaction terminating layer.
Another similar method described by W. R. Hargreaves in U.S. Pat. No. 4,868,130, describes the use of an imiscible boundary effect to separate bound from unbound labelled components in an immunological reaction mixture. The specific binding reaction occurs in a solution which is immiscible with an underlying "primary layer". The resulting labelled analyte complex is sufficiently different from the unbound label, e.g. bound to particles, such that when a force is applied to the labelled analyte complex it is drawn into the primary layer, leaving the unbound label behind in the reaction mixture. If the label is fluorescent, then Hargreaves uses side-excitation in the bottom region of the primary layer to prevent excitation of the free label in the upper aqueous layer, the detector being at a right angle thereto.